TGF-β receptor type I (TGF-βRI) kinase (IC50 = 56 nM)

在 SK-Sora、HepG2 和 Hep3B 细胞系中，galunisertib (LY2157299)（0.1、1、10 和 100 μM）以剂量依赖性方式在一定程度上增强 Bay 43-9006；然而，在 JHH6、SK-HEP1 或 HuH7 细胞系中未观察到这种效应[2]。Galunisterib（LY2157299）是一种选择性ATP模拟抑制剂，用于抑制TGF-β受体（TβR）-I的激活，目前正在肝细胞癌（HCC）患者的临床研究中。我们的研究探讨了galunisterib在HCC细胞系中的体外作用和对患者样本的离体作用。在HepG2、Hep3B、Huh7、JHH6和SK-HEP1细胞以及耐受索拉非尼（SK Sora）和舒尼替尼（SK Suni）的SK-HEP1-衍生细胞中评估了Galunisterib。TGF-β对所有HCC细胞系的外源性刺激产生了p-Smad2和p-Smad3的下游激活，在微摩尔浓度下，galunisterib治疗可有效抑制这些激活。尽管抗增殖作用有限，但galunisterib具有强大的抗侵袭特性。13名接受手术切除的HCC患者的肿瘤切片在体外暴露于1µM和10µM的galunisterib、5µM的索拉非尼或两种药物的组合48小时。Galunisterib而非索拉非尼降低了TGF-β下游的p-Smad2/3信号传导。Galunisterib和索拉非尼暴露样本的免疫组织化学分析显示，增殖标志物Ki67显著降低，凋亡标志物caspase-3增加。结合使用，galunisterib通过抑制增殖和增加凋亡有效地增强了索拉非尼的作用。

人类异种移植物 Calu6（非小细胞肺癌）和 MX1（乳腺癌）的皮下植入是在裸鼠中进行的。当以 75 mg/kg 的剂量口服时，Galunisertib (LY2157299) 会导致两种细胞系的 pSmad 降低 70%。给药后约 6 小时，pSmad 恢复至基线的 80% [3]。

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将人异种移植物Calu6（非小细胞肺癌癌症）和MX1（癌症）皮下植入裸鼠体内，并口服新型I型受体TGF-β激酶拮抗剂LY2157299。LY2157299的血浆水平、肿瘤中磷酸化Smad2,3（pSmad）的百分比和肿瘤大小用于建立半机械药代动力学/药效学模型。使用间接反应模型将血浆浓度与pSmad联系起来。该模型预测pSmad的完全抑制和快速周转率[t（1/2）（min）=18.6（Calu6）和32.0（MX1）]。使用两个信号转导室将肿瘤生长抑制与pSmad联系起来，这两个信号传导室的特征是平均信号传播时间，Calu6和MX1的估计值分别为6.17天和28.7天。该模型提供了一种生成实验假设的工具，以深入了解与TGF-β膜受体I型相关的信号转导机制。[3]

最近，激酶抑制剂对纤维化疾病显示出巨大的潜力，特别是转化生长因子β受体（TGF-βR）被发现是硬皮病治疗的一个新的有前景的靶点。在目前的研究中，我们提出可以利用现有的大量激酶抑制剂来抑制TGF-βR，从而抑制硬皮病。在这方面，我们开发了一种建模方案，系统地分析了169种市售激酶抑制剂对TGF-βR的抑制活性，从中选择了五种有前景的候选药物，并使用标准激酶测定方案进行了测试。因此，两种分子实体，即PKB抑制剂MK-2206和mTOR C1/C2抑制剂AZD8055，在与TGF-βR结合时显示出高效力，IC50值分别为97和86 nM，接近最近开发的TGF-βR选择性抑制剂SB525334和galunisterib/LY2157299（IC50分别为14.3和56 nM）。我们还进行了原子分子动力学模拟和后分子力学/泊松-玻尔兹曼表面积分析，以剖析TGF-βR激酶结构域与这些强效化合物之间分子间相互作用的结构基础和能量特性，突出了非同源TGF-βR抑制剂复合物紧密堆积界面上的密集非结合网络[1]。

细胞毒性试验[2]
使用MTT法（3-[4,5-二甲基噻唑-2-基]-2,5-二苯基溴化四唑）测定细胞存活率。黄色水溶性四氮唑MTT转化为紫色不溶性甲酰胺是由线粒体脱氢酶催化的，用于估算活细胞的数量。简而言之，细胞以2×103个细胞/孔的密度接种在96孔组织培养板上。药物暴露后，将细胞与0.4 mg/mL MTT在37°C下孵育4小时。孵育后，丢弃上清液，将不溶性甲赞沉淀物溶解在0.1mL DMSO中，并使用酶标仪在560nm处测量吸光度。分别使用含有未经处理的细胞或不含细胞的含药物培养基的孔作为阳性对照和阴性对照。对于增殖试验，每天进行MTT试验，以确定未经治疗的对照组和galunispertib治疗组中活细胞的数量。

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活体外组织分析（TIPCAN®）[2]
在HCC患者新鲜切除的肿瘤上测试了galunisterib的效果，这些肿瘤可以在特定的培养基和大气条件下进行活培养，具体取决于外科可用的肿瘤切除。在医院病理学家进行病理评估后，使用Tissue Slicer®仪器将肿瘤样本临时切成300μm厚的切片，并在37°C下在William’s E培养基中“活”培养，在常氧条件下补充内部专有的专用成分，包括胎牛血清、葡萄糖、庆大霉素和HEPES。使用组织切片技术制备样品，并用1和10μM的galunispertib或5μM的索拉非尼处理24至72小时。处理24至72小时后，将外植的HCC石蜡包埋并评估所选标志物的表达。测试包括评估癌症细胞增殖（MIB1/Ki67）、死亡（活性胱天蛋白酶-3）和细胞信号传导的几种变化（磷酸激酶）。组织质量由病理学家评估。如果随着时间的推移，组织的完整性没有得到维持（坏死诱导率>20%），则丢弃组织。

PK/PD experiments[3]
Calu6[3]
LY2157299 was given orally as a single dose (data from eight independent studies were combined) or in a multiple dosing design (one study). The value of the dose levels given in a single dose manner was 10 (n = 3), 30 (n = 8), 50 (n = 26), 75 (n = 69), 100 (n = 3), 150 (n = 21) and 300 (n = 3) mg/kg. Animals were sacrificed at the following times: 0.5, 1, 1.5, 2, 4, 8 and 16 h after administration, then the tumour was removed and blood was recovered. In the multiple dosing study, LY2157299 was administered twice a day (bid) at the dose of 75 mg/kg every 12 h for 20 consecutive days to 31 mice. Animals were sacrificed at 2 h after the last administration at days 10, 15, 20 and 25, and the tumour was removed for pSmad determination and the blood was recovered for determination of drug levels in plasma. [3]

MX1[3]
Twelve mice involved in a single study were treated with a single 75 mg/kg dose of LY2157299. Animals were sacrificed at 0.5, 1, 2, 4 and 16 h after drug administration, tumours were removed and the blood was collected. [3]

Determination of LY2157299 in plasma[3]
Venous blood samples (1 ml) was drawn into sodium-heparinised tubes for measurement of LY2157299. Plasma samples were analysed using a validated method involving protein precipitation with turbo ion spray LC/MS/MS detection. The validated range of measurement in plasma was 5–1000 ng/ml (a 50-fold dilution was validated to demonstrate the ability of the assay to analyse samples at higher concentrations). The value of the limit of quantification of the assay was 1.14 ng/ml. The accuracy of the assay was <15% and the intra and interassay coefficients of variation were less than 10%.[3]

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Tumour growth experiments[3]
Calu6[3]
Data from two studies are presented. The first available data came from a study where 20 mice were treated bid with either saline (control group; n = 10) or 75 mg/kg of LY2157299 (treated group; n = 10) for 20 consecutive days. Tumour size was measured every 4–6 days for one month after the first drug administration and afterwards the animals were sacrificed. The data from this study were used to develop the tumour growth model (index dataset). Later, data from a second study also became available and were used for model validation purposes (test dataset). Seventy-six mice were treated bid with either saline (control group; n = 36) or 75 mg/kg of LY2157299 for 10 (n = 10) 15 (n = 10) or 20 (n = 20) consecutive days. Tumour size was measured once a week for one month.

MX1[3]
Data were obtained from a single study where mice were treated three times a day with either saline (control group, n = 10) or 75 mg/kg of LY2157299 (treated group; n = 10) for 20 consecutive days. Tumour size was measured every 3–4 days for one month after the first drug administration and afterwards the animals were killed.

Dissolved in DMSO and diluted in saline; 75 mg/kg/day; oral gavage

Nude mice implanted subcutaneously with Calu6 or MX1 cells

Pharmacokinetic parameters were determined for patients administered galunisertib during the first 14 days of the 28-day intermittent treatment cycle (2 weeks on/2 weeks off schedule). The PK profile of galunisertib was characterized by rapid absorption, with median t max ranging from 0.5 to 2 h following oral dosing with 80 or 150 mg BID (Fig. 2). At steady state, on Day 14 in Cycle 1, the mean t 1/2 was 8.90 h and the mean CLss/F and Vz,ss/F during the terminal phase were 30.2 L/h and 388 L, respectively, for 150 mg BID (Table 3). Although the number of patients in the 2 cohorts was small and imbalanced (Cohort 1, n = 3; Cohort 2, n = 9), high interpatient variability for galunisertib exposure [AUC(0−48) coefficient of variation (CV) %] was observed (Cohort 1 CV % = 35 %; Cohort 2 CV % = 88 %). [5]

Galunisertib administered to 12 Japanese patients with advanced solid tumors was well tolerated and had a favorable safety profile; no DLTs or cardiovascular toxicities were reported. Dose escalation was successfully performed within the 2 dosing cohorts (80 and 150 mg BID) and galunisertib exposure data confirmed that exposure could be maintained within the predefined therapeutic window for the majority of patients during treatment with galunisertib. All patients completed at least one cycle of galunisertib treatment before discontinuing due to disease progression; no patients had a clinical response to treatment, however, two patients had stable disease. [5]

The favorable tolerability and safety profile of 80 and 150 mg BID doses of galunisertib in Japanese patients was confirmed based on the TEAE profile reported during the study. Overall, there were no CTCAE Grade ≥3 study drug-related toxicities reported. Possible drug-related TEAEs included two patients with increased BNP levels, two patients with leukopenia, and two patients with rash. The two patients with increased BNP (Grade 1) did not experience any cardiotoxicities, and no febrile neutropenia was reported for any patient. Possible study drug-related leukopenia (n = 3 events) was also reported in the FHD study in patients who received a combination of galunisertib and lomustine; however, causality could not be specifically attributed to either drug. Therefore, it is unclear if the reported leukopenia was related to galunisertib treatment.

[1]. Targeting the TGF-β receptor with kinase inhibitors for scleroderma therapy. Arch Pharm (Weinheim). 2014 Sep;347(9):609-15.

[2]. Effects of TGF-beta signalling inhibition with galunisertib (LY2157299) in hepatocellular carcinoma models and in ex vivo whole tumor tissue samples from patients. Oncotarget. 2015 Aug 28;6(25):21614-27.

[3]. Semi-mechanistic modelling of the tumour growth inhibitory effects of LY2157299, a new type I receptor TGF-beta kinase antagonist, in mice. Eur J Cancer. 2008 Jan;44(1):142-50.

[4]. Clinical development of galunisertib (LY2157299 monohydrate), a small molecule inhibitor of transforming growth factor-beta signaling pathway. Drug Des Devel Ther. 2015 Aug 10;9:4479-99.

[5]. Phase 1 study of galunisertib, a TGF-beta receptor I kinase inhibitor, in Japanese patients with advanced solid tumors. Cancer Chemother Pharmacol . 2015 Dec;76(6):1143-52.

LY-2157299 is a pyrrolopyrazole that is 5,6-dihydro-4H-pyrrolo[1,2-b]pyrazole which is substituted at positions 2 and 3 by 6-methylpyridin-2-yl and 6-(aminocarbonyl)quinolin-4-yl groups, respectively. A Transforming growth factor-betaRI (TGF-betaRI) kinase inhibitor, it blocks TGF-beta-mediated tumor growth in glioblastoma. It has a role as a TGFbeta receptor antagonist and an antineoplastic agent. It is a member of quinolines, a pyrrolopyrazole, a member of methylpyridines, an aromatic amide and a monocarboxylic acid amide.
Galunisertib has been used in trials studying the basic science and treatment of Glioma, Neoplasms, Solid Tumor, GLIOBLASTOMA, and Prostate Cancer, among others.
Galunisertib is an orally available, small molecule antagonist of the tyrosine kinase transforming growth factor-beta (TGF-b) receptor type 1 (TGFBR1), with potential antineoplastic activity. Upon administration, galunisertib specifically targets and binds to the kinase domain of TGFBR1, thereby preventing the activation of TGF-b-mediated signaling pathways. This may inhibit the proliferation of TGF-b-overexpressing tumor cells. Dysregulation of the TGF-b signaling pathway is seen in a number of cancers and is associated with increased cancer cell proliferation, migration, invasion and tumor progression.

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Galunisertib (LY2157299) is a selective ATP-mimetic inhibitor of TGF-β receptor (TβR)-I activation currently under clinical investigation in hepatocellular carcinoma (HCC) patients. Our study explored the effects of galunisertib in vitro in HCC cell lines and ex vivo on patient samples. Galunisertib was evaluated in HepG2, Hep3B, Huh7, JHH6 and SK-HEP1 cells as well as in SK-HEP1-derived cells tolerant to sorafenib (SK-Sora) and sunitinib (SK-Suni). Exogenous stimulation of all HCC cell lines with TGF-β yielded downstream activation of p-Smad2 and p-Smad3 that was potently inhibited with galunisertib treatment at micromolar concentrations. Despite limited antiproliferative effects, galunisertib yielded potent anti-invasive properties. Tumor slices from 13 patients with HCC surgically resected were exposed ex vivo to 1 µM and 10 µM galunisertib, 5 µM sorafenib or a combination of both drugs for 48 hours. Galunisertib but not sorafenib decreased p-Smad2/3 downstream TGF-β signaling. Immunohistochemistry analysis of galunisertib and sorafenib-exposed samples showed a significant decrease of the proliferative marker Ki67 and increase of the apoptotic marker caspase-3. In combination, galunisertib potentiated the effect of sorafenib efficiently by inhibiting proliferation and increasing apoptosis. Our data suggest that galunisertib may be active in patients with HCC and could potentiate the effects of sorafenib.[2]

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Human xenografts Calu6 (non-small cell lung cancer) and MX1 (breast cancer) were implanted subcutaneously in nude mice and LY2157299, a new type I receptor TGF-beta kinase antagonist, was administered orally. Plasma levels of LY2157299, percentage of phosphorylated Smad2,3 (pSmad) in tumour, and tumour size were used to establish a semi-mechanistic pharmacokinetic/pharmacodynamic model. An indirect response model was used to relate plasma concentrations with pSmad. The model predicts complete inhibition of pSmad and rapid turnover rates [t(1/2) (min)=18.6 (Calu6) and 32.0 (MX1)]. Tumour growth inhibition was linked to pSmad using two signal transduction compartments characterised by a mean signal propagation time with estimated values of 6.17 and 28.7 days for Calu6 and MX1, respectively. The model provides a tool to generate experimental hypothesis to gain insights into the mechanisms of signal transduction associated to the TGF-beta membrane receptor type I.[3]

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Transforming growth factor-beta (TGF-β) signaling regulates a wide range of biological processes. TGF-β plays an important role in tumorigenesis and contributes to the hallmarks of cancer, including tumor proliferation, invasion and metastasis, inflammation, angiogenesis, and escape of immune surveillance. There are several pharmacological approaches to block TGF-β signaling, such as monoclonal antibodies, vaccines, antisense oligonucleotides, and small molecule inhibitors. Galunisertib (LY2157299 monohydrate) is an oral small molecule inhibitor of the TGF-β receptor I kinase that specifically downregulates the phosphorylation of SMAD2, abrogating activation of the canonical pathway. Furthermore, galunisertib has antitumor activity in tumor-bearing animal models such as breast, colon, lung cancers, and hepatocellular carcinoma. Continuous long-term exposure to galunisertib caused cardiac toxicities in animals requiring adoption of a pharmacokinetic/pharmacodynamic-based dosing strategy to allow further development. The use of such a pharmacokinetic/pharmacodynamic model defined a therapeutic window with an appropriate safety profile that enabled the clinical investigation of galunisertib. These efforts resulted in an intermittent dosing regimen (14 days on/14 days off, on a 28-day cycle) of galunisertib for all ongoing trials. Galunisertib is being investigated either as monotherapy or in combination with standard antitumor regimens (including nivolumab) in patients with cancer with high unmet medical needs such as glioblastoma, pancreatic cancer, and hepatocellular carcinoma. The present review summarizes the past and current experiences with different pharmacological treatments that enabled galunisertib to be investigated in patients.[4]

C22H19N5O

369.42

369.158

C, 71.53; H, 5.18; N, 18.96; O, 4.33

700874-72-2

700874-72-2;924898-09-9 (hydrate);

10090485

White to yellow solid powder

1.4±0.1 g/cm3

619.0±55.0 °C at 760 mmHg

328.2±31.5 °C

0.0±1.8 mmHg at 25°C

1.751

1.73

86.69

1

4

3

28

585

0

IVRXNBXKWIJUQB-UHFFFAOYSA-N

InChI=1S/C22H19N5O/c1-13-4-2-5-18(25-13)21-20(19-6-3-11-27(19)26-21)15-9-10-24-17-8-7-14(22(23)28)12-16(15)17/h2,4-5,7-10,12H,3,6,11H2,1H3,(H2,23,28)

4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline-6-carboxamide

LY2157299; LY2157299; 4-(2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinoline-6-carboxamide; UNII-3OKH1W5LZE; ly2157299(galunisertib); LY 2157299

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

配方 1 中的溶解度: ≥ 5.75 mg/mL (15.56 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (5.63 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.08 mg/mL (5.63 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 4 中的溶解度: ≥ 2.08 mg/mL (5.63 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，您可以将 100 μL 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 5 中的溶解度： 2% DMSO+30% PEG 300+ddH2O:5 mg/mL

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。